CN103215292A - Human Pcid2 protein soluble expression method, anti-human Pcid2 protein monoclonal antibody 2D7-F11, and hybridoma cell line secreting antibody - Google Patents
Human Pcid2 protein soluble expression method, anti-human Pcid2 protein monoclonal antibody 2D7-F11, and hybridoma cell line secreting antibody Download PDFInfo
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Abstract
The invention provides a human Pcid2 protein soluble expression method, an anti-human Pcid2 protein monoclonal antibody obtained through using a soluble Pcid2 recombinant protein as an antigen of immune mice, and a hybridoma cell line secreting the antibody. The monoclonal antibody has a strong positive reaction to the recombinant human Pcid2 protein and cells expressing a human natural Pcid2 protein, and has no cross reactions to other proteins. The invention also provides a kit including the monoclonal antibody, and a method for detecting the endogenous Pcid2 content through using the monoclonal antibody.
Description
Technical field
The present invention relates to the recombinant expressed and monoclonal antibody field of albumen, more specifically, it is recombinant expressed to the present invention relates to the proteic solubility of anti-people Pcid2, and utilizing the monoclonal antibody 2D7-F11 of the Pcid2 albumen of solubility expression for antigen prepd, it is that 2D7-F11 produces by mouse hybridoma cell.
Background technology
Pcid2 (PCI domain containing 2) albumen is the conservative a kind of albumen of eukaryotic cell camber.Its typical constitutional features is to have one section PCI (proteasome, COP9 signalosome, initiation factor 3) structural domain in its sequence.The PCI structural domain is a class functional domain that extensively exists in Eukaryotic proteasome, COP9 complex body and transcription initiation factor 3 complex bodys.Generally be positioned at the C end of associated protein, about 200 amino acid are commonly used to mediate albumen and albumen interphase interaction in the protein complexes.The albumen that contains the PCI structural domain can appear in the big multiprotein complex usually, for example 26S proteasome, COP9 signal complex body, transcription initiation factor 3 complex bodys.
People Pcid2 albumen contains 399 amino acid altogether, and its C end is the PCI structural domain.Pcid2 albumen has the conservative property of height in eukaryote.The yeast homologous protein Thp1 of Pcid2 is present in the TREX-2/Sac3-Thp1-Sus1-Cdc31 complex body, and the nucleopore transportation of the nucleopore location (" gene gating ") of this complex body and transcriptional activation gene, mRNA is closely related.Report simultaneously points out that the Pcid2 albumen in fruit bat has identical character, can shuttle back and forth in nucleus and endochylema, transports closely related with the nuclear that goes out of mRNA.
Have report to point out recently, the survival of Pcid2 and cell, breed closely related.Embryonic death can take place in the Pcid2 knock out mice.After utilizing the RNA perturbation technique to suppress the interior proteic expression of Pcid2 of cell, the propagation of cell is blocked substantially.Other has research to point out, in the mouse that the Pcid2 conditionality knocks out, pounds out the Pcid2 albumen in the B cell specifically, can cause the apoptosis in the B cell development process, finally causes the decline of B cell number.
Pcid2 albumen is also relevant with atomization and the self of stem cell.There is the investigator to utilize large-scale RNA to disturb screening strategy, obtained comprising 148 genes relevant of Pcid2 with differentiation of stem cells and self.Further experimental result has been proved conclusively this discovery.With the index of alkaline phosphatase as cytodifferentiation, to find Pcid2 is carried out after RNA disturbs, the dye level of alkaline phosphatase descends, and indicates that differentiation has taken place stem cell.
Also there is report to point out recently, in more refractory three negative breast cancer of a class (Triple negative breast cancer), finds that in outgrowth tumor tissues the unusual amplification of copy number can take place the Pcid2 gene.This is indicating that Pcid2 albumen may offer help to the diagnosis and the treatment of three negative breast cancer.
The potential use of Pcid2 albumen monoclonal antibody comprises: be used for detecting content and the distribution of stem cell tissue Pcid2, as the indication of differentiation of stem cells 1.; 2. content and the distribution of Pcid2 in the detection tumor tissues is as the index of tumour generation and development.
Summary of the invention
The purpose of this invention is to provide a kind of monoclonal antibody 2D7-F11 that expresses proteic method of solubility Pcid2 and anti-people Pcid2.
Aspect first, the invention provides the proteic method of a kind of expression solubility Pcid2, the interaction protein Dss1 that it is characterized in that utilizing Pcid2 promotes the solubility expression of Pcid2 with the coexpression of Pcid2, described method comprises:
(a) obtain the proteic nucleic acid sequence encoding of Pcid2 albumen and Dss1 respectively;
(b) Pcid2 albumen and the Dss1 encoding histone nucleotide sequence that obtains in (a) inserted in the carrier that is fit to;
(c) express under the condition that the carrier that structure in (b) is obtained is being suitable for expressing in the host who is fit to.
In one embodiment, the proteic nucleic acid sequence encoding of described Pcid2 is SEQ ID NO:1.In one embodiment, the proteic nucleic acid sequence encoding of described Dss1 is SEQ ID NO:2.In one embodiment, described carrier is a prokaryotic expression carrier, is in particular pETDuet-1.In one embodiment, described host is intestinal bacteria (Escherichia coli) bacterial strains.In one embodiment, described bacterial strain is an intestinal bacteria BL-21 bacterial strain.
In aspect second, the invention provides a kind of soluble recombined human Pcid2/Dss1 albumen, its method by first aspect produces.In one embodiment, the proteic nucleic acid sequence encoding of described Pcid2 is SEQ ID NO:1.In one embodiment, the proteic nucleic acid sequence encoding of described Dss1 is SEQ ID NO:2.
In aspect the 3rd, the invention provides second described people in aspect of a kind of the present invention of utilization proteic monoclonal antibody of anti-people Pcid2 that Pcid2 albumen produces for antigen of recombinating.In one embodiment, monoclonal antibody is that the mouse hybridoma cell of CGMCC No.5014 is that 2D7-F11 produces by deposit number.
In aspect the 4th, the invention provides a kind of hybridoma cell line, its deposit number is CGMCC No.5014.
In aspect the 5th, the invention provides the test kit that is used to detect the Pcid2 protein content, it comprises second the described reorganization in aspect Pcid2/Dss1 albumen and/or the 3rd the described monoclonal antibody in aspect.
In aspect the 6th, the invention provides according to the application of the 3rd the described monoclonal antibody in aspect in the co-immunoprecipitation method of the immunohistochemical methods method that detects the Pcid2 tissue distribution, the immunofluorescence method that detects the Pcid2 cell distribution and detection Pcid2 interaction protein.
The present invention is an antigen with solubility Pcid2 albumen, by immune mouse and follow-up monoclonal antibody screening preparation process, has obtained the proteic monoclonal antibody at Pcid2.Further the antibody function check shows that this monoclonal antibody 2D7-F11 can discern recombinant expressed Pcid2 albumen, also can discern the Pcid2 albumen of endogenous expression.This monoclonal antibody can be applied to aspects such as ELISA, Western Blot, immunoprecipitation, immunofluorescence.There is not non-specific recognition with other albumen in the cell.
Specifically, the invention provides following content:
1) the proteic method of a kind of solubility expression reorganization Pcid2, it passes through the proteic interaction protein Dss1 of coexpression Pcid2, thereby stablizes the proteic structure of Pcid2, and Pcid2 albumen can be existed in external solubility.
2) the proteic monoclonal antibody 2D7-F11 of a kind of anti-people Pcid2, it is produced by mouse hybridoma cell 2D7-F11 (CGMCC No.5014).Can be used in experiments such as ELISA, immunoblotting, immunoprecipitation, immunofluorescence and immunohistochemistry detects.
3) a kind of hybridoma that produces the proteic monoclonal antibody 2D7-F11 of anti-people Pcid2 is characterized in that, it is that preserving number is that the mouse hybridoma cell of CGMCC No.5014 is 2D7-F11.
4) be used to detect the test kit of Pcid2 protein content, it comprises above 1), 2) described solubility Pcid2 albumen and monoclonal antibody 2D7-F11 thereof.The preparation of test kit can be undertaken by method known in those skilled in the art.
5) according to above 2) antibody or 4) test kit, wherein detect proteic content of the Pcid2 that obtains and distribution situation, can be used as the sign and the outgrowth sign of three negative breast cancer of development of stem cells differentiation.
Description of drawings:
Fig. 1 .pETDuet-1 carrier collection of illustrative plates.
Fig. 2 .Western blotting detects the Pcid2 albumen in the MCF-7 cell.
Fig. 3. immunofluorescence detects Pcid2 albumen among the MCF-7.
Pcid2 albumen in Fig. 4 .2D7-F11 immunoprecipitation MCF-7 cell pyrolysis liquid (left side: IgG contrast, the right side: Pcid2 mAb).
Fig. 5. immunohistochemical methods detects Pcid2 expression in breast cancer tissue's section.
Embodiment
Can there be high expression level in Pcid2 albumen stem cell, has the critical function of keeping stem cell self (self-renewal) ability.Experiment shows, by RNA interferential method, reduces the proteic level of Pcid2 in the stem cell, can cause the decline of stem cell self ability, and the appearance of differentiation shows as the unusual of alkaline phosphatase staining.Similarly report is also pointed out, Pcid2 albumen may be relevant with the differentiation of B cells whose development, and the Pcid2 albumen of conditionality knock-out mice B cell expressing can finally cause the decline of B cell number in the peripheral blood.Further experimental results show that some the B cell precursor cells in the B cell differentiation procedure, the minimizing of cell number has appearred in the influence that knocked out by Pcid2 albumen, and this explanation Pcid2 is bringing into play important effect in B cells whose development atomization.In addition, Pcid2 albumen is also closely related with apoptosis and cell cycle regulating.Same piece of writing report points out that in the B cell that Pcid2 albumen knocks out, the activity of Caspase3 improves greatly, causes a large amount of B cells the apoptosis characteristic to occur.In addition, Pcid2 can influence the expression level of MAD2 in the cell, and the RNA experiment shows that Pcid2 knocks out in the cell, and the level of MAD2 mRNA obviously descends in the endochylema, finally causes the downward modulation of MAD2 protein expression level.MAD2 is an important albumen of regulating the cell cycle in the mitotic division process, and its function guarantees the correct formation of spindle body in the cell cycle process.The proteic wrong downward modulation of MAD2 can cause the cell cycle to be stagnated in the mitotic division process, shows clearly multinuclear characteristic thereby make Pcid2 knock out cell.In addition, there is report to point out recently again, in three negative breast cancer hyperplasia processes, the amplification of copy number appears in the Pcid2 gene, this shows Pcid2 albumen, and the pathological process with three negative breast cancer is relevant probably, thereby may provide a kind of molecular indexes for the pathology detection and the healing approach of three negative breast cancer.Molecular mechanism about the Pcid2 function also is not very clear.Its homologous protein studies show that, the Pcid2 homologous protein of yeast and fruit bat all with mRNA to go out the nuclear transportation closely related, the disappearance of its homologous protein can cause assembling in the nuclear of mRNA.In addition, other albumen with PCI structural domain can form big complex body protein structure by the PCI structural domain usually, be present in proteasome, COP9 complex body, eukaryotic translation initiation factor complex body 3 and some other complex structures the biological function that performance is complicated.People's Pcid2 albumen has similar molecular mechanism with its yeast and fruit bat homologous protein and other albumen with PCI structural domain probably, and its complicated function is needed further investigation badly.But, recombinant expressed for this proteic solubility at present without any report.Present known antibody can only be at the recombinant protein of the inclusion body form of Western Blot level identification sex change, can't detect for the Pcid2 albumen of endogenous expression in the born of the same parents.
The present invention includes: a kind of method of solubility expression Pcid2 recombinant protein, it obtains by Pcid2 albumen and its interaction protein Dss1 coexpression.The proteic antibody 2D7-F11 of one species specific anti-people Pcid2, it is that 2D7-F11 produces by mouse hybridoma cell.This antibody experimentally has widely at immunologic function to be used.With reorganization Pcid2 protein immunization Balb/C mouse, use the hybridoma technology, prepared the cell strain 2D7-F11 that a strain can stably excreting specific anti-human Pcid2 protein monoclonal antibody.
The method of a kind of solubility expression Pcid2 recombinant protein among the present invention is to obtain by coexpression Pcid2 and its interaction protein Dss1.The Pcid2 single expression all exists with the inclusion body form, has only with the Dss1 coexpression to form correct conformation, is present in the supernatant.
A kind of positive hybridoma cell among the present invention is anti-people Pcid2 protein monoclonal antibody hybridoma cell strain 2D7-F11, this hybridoma cell strain is preserved in the common micro-organisms center (CGMCC of China Committee for Culture Collection of Microorganisms on June 30th, 2011, China, Beijing), preserving number is CGMCC No.5014.
The experiment of anti-people Pcid2 protein monoclonal antibody 2D7-F11 among the present invention shows that the Pcid2 albumen of 2D7-F11 and recombinant human Pcid2 albumen and cell or tissue endogenous expression is strong positive reaction, but does not have positive reaction with the Dss1 albumen of coexpression.There is not cross reaction with other albumen in the cell.
The proteic monoclonal antibody 2D7-F11 of anti-people Pcid2 has following characteristics and performance among the present invention: in (1) ELISA experiment with reorganization Pcid2 albumen and cell pyrolysis liquid in Pcid2 albumen be strong positive reaction; (2) can discern the reorganization Pcid2 albumen of sex change and the proteic band of Pcid2 of endogenous expression in the Western Blot experiment; (3) immunoprecipitation experiment can specificity in conjunction with endogenic Pcid2 albumen in reorganization and the cell pyrolysis liquid; (4) immunofluorescence experiment can be discerned the Pcid2 albumen in the fixed cell; (5) immunohistochemical assay can be discerned the Pcid2 albumen in the fixed tissue slices.
The preparation of solubility Pcid2 recombinant protein and purifying
(1) expression plasmid makes up
Usually with the expression of recombinant proteins difficulty or ease, utilizing colibacillary prokaryotic expression strategy is the most simple protein expression strategy.We successfully utilize prokaryotic expression to obtain the Pcid2 albumen of solubility.At first, in order to express Pcid2 albumen and interaction protein Dss1 thereof, we utilize round pcr to increase from cDNAs and obtain these two sequences (NCBI sequence number NP_001120675.1, NP_006295.1), on passing through restriction enzyme digestion and being connected the commercialization prokaryotic expression carrier pETDuet-1 that is building up to Novagen, wherein Pcid2 albumen has the His label, the purifying that is used for recombinant protein, and Dss1 is not with any label.The pDuet plasmid can guarantee two proteic while accurate translations.
The expression vector establishment process:
The Pcid2 nucleotide sequence:
DNA?Sequence?Open?Reading?Frame：82?to?1281(SEQ?ID?NO：1)
NM_018356.2
AGACGGACCG?AGGGCCGGAA?GTGACGCCAG?CTGGCCCGCT?TGAGGCGTAG?GGGGTGGCGC?TCTCCGTTCG
GCGGCGCTCC?CATGGCGCAC?ATTACCATTA?ACCAGTACCT?GCAGCAGGTG?TACGAAGCCA?TCGACAGCAG
AGATGGAGCA?TCTTGTGCAG?AGTTGGTGTC?TTTTAAACAT?CCTCATGTTG?CAAACCCACG?ACTTCAAATG
GCCTCTCCAG?AGGAGAAGTG?TCAACAAGTC?TTGGAACCCC?CTTATGATGA?AATGTTTGCA?GCTCATTTAA
GGTGCACTTA?TGCAGTGGGG?AATCATGACT?TCATAGAGGC?ATACAAGTGC?CAGACCGTGA?TAGTCCAATC
ATTCTTGCGA?GCATTGCAGG?CCCACAAAGA?AGAAAACTGG?GCTCTGCCTG?TCATGTATGC?AGTAGCGCTT
GACCTTCGAG?TGTTTGCCAA?TAATGCAGAT?CAACAGTTGG?TAAAGAAAGG?AAAAAGCAAA?GTTGGGGACA
TGTTGGAAAA?AGCAGCAGAG?TTACTGATGA?GCTGTTTCCG?GGTCTGTGCC?AGCGACACCC?GTGCTGGTAT
AGAGGACTCT?AAGAAGTGGG?GCATGCTGTT?TCTGGTGAAC?CAGCTGTTTA?AAATCTACTT?CAAGATCAAC
AAACTCCATT?TATGTAAACC?CCTAATTAGA?GCAATTGACA?GCTCAAACCT?GAAAGACGAT?TACAGCACTG
CACAGAGAGT?AACATACAAA?TACTACGTTG?GACGCAAGGC?TATGTTTGAC?AGCGATTTTA?AGCAAGCTGA
GGAGTACCTG?TCATTTGCCT?TTGAGCATTG?TCACCGTTCT?AGTCAGAAGA?ACAAAAGGAT?GATTCTGATC
TATTTGCTTC?CAGTAAAAAT?GCTATTGGGT?CACATGCCCA?CTGTGGAGCT?CCTGAAAAAG?TATCACCTGA
TGCAGTTTGC?GGAAGTAACC?AGAGCTGTGA?GCGAGGGCAA?CCTGCTGCTG?CTGCACGAGG?CGCTGGCGAA
GCACGAGGCC?TTCTTCATTC?GCTGCGGAAT?CTTCCTCATC?CTGGAGAAGC?TGAAGATCAT?CACCTACAGG
AACCTCTTTA?AGAAAGTGTA?TTTGTTACTG?AAAACACACC?AGCTGTCTCT?GGATGCTTTT?CTGGTTGCCT
TGAAGTTCAT?GCAGGTGGAG?GACGTGGACA?TTGACGAAGT?TCAGTGTATT?CTGGCTAACT?TGATATACAT
GGGACACGTC?AAAGGCTACA?TATCGCATCA?GCATCAGAAG?CTGGTGGTCA?GCAAGCAGAA?CCCATTTCCT
CCCCTGTCCA?CGGTGTGTTG?AAAGTACACG?GAGCCCCGAG?GACGGACTCG?GCTGGTTCTG?GAGTCTTTGT
GAGACTTCTT?TGAAGGAGGC?TTTGCGTGAA?GGCTGCTCGG?CTCACTTTTC?CTAAGTGTGG?TTCCTGAAGG
CTGTCTTTGT?AACTTTTTGT?AGTTCTTTGT?GTAAAAAGCG?TATTCTGAAT?TTATACACAT?GGCATGTT
CT
TCATTATATC?TTCCAGGATA?CATCTATTTT?TATATATTAA?ATTTGAATGT?GTTATCAAAA?TGCTTGGTTA
ACTTAAGGCA?CCTTTTTAAA?AGCAGAATTT?AATTTGATTT?AAATTTTCCA?GATTTTATAG?CTTGCCTGTA
TGGATGCTCC?TCAATTTATG?ATAGGGTTAC?ATCCCAATAA?ACTTATTTTA?TTTGCCTTTG?CAAAAAAAAA
AAAAAAAAAA?AAAAAAAAAA?AAAAAAAAAA?AAAAAAAAAA?AAAAAAAAAA?AAAAAAAAAA?AAAAAAAAAA?AAA
The Dss1 nucleotide sequence:
DNA?Sequence?Open?Reading?Frame：129?to?341(SEQ?ID?NO：2)
NM_006304.1
ATTCTTTCCC?CAAGTCTCTA?TGGTAGCGTC?AGCGTCGGAG?GCGGTAGTGA?CGGTGGCGTT?TCCTTGAGGA
AGAGTGAGGG?TTCCAACTTT?TCTGCTTATC?TGGGAGGTGT?TGGGCGCGGA?CAGTCGAGAT?GTCAGAGAAA
AA
GCAGCCGG?TAGACTTAGG?TCTGTTAGAG?GAAGACGACG?AGTTTGAA
GA?GTTCCCTGCC?GAAGACTGGG
CTGGCTTAGA?TGAAGATGAA?GATGCACATG?TCTGGGAGGA?TAATTGGGAT?GATGACAATG?TAGAGGATGA
CTTCTCTAAT?CAGTTACGAG?CTGAACTAGA?GAAACATGGT?TATAAGATGG?AGACTTGATA?GCATCCAGAA
GAAGTGTTGA?AGTAACCTAA?ACTTGACCTG?CTTAATACAT?TCTAGGGCAG?AGAACCCAGG?ATGGGACACT
AAAAAAATGT?GTTTATTTCA?TTATCTGCTT?GGATTTATTT?GTGTTTTTGT?AACACAAAAA?ATAAATGTTT
TGATATAAAA?AAAAAAAAA
Utilize PCR that Pcid2 (SEQ ID NO:1), Dss1 (SEQ ID NO:2) amplification is come out and add double enzyme site at the sequence two ends (Pcid2 two ends restriction enzyme site is EcoRI, HindIII; Dss1 two ends restriction enzyme site is NdeI, XhoI), MCS1, the MCS2 position of above-mentioned two sections sequences being inserted pETDuet-1 carrier (pETDuet-1 carrier collection of illustrative plates is seen Fig. 1) respectively.
(2) Pcid2 protein expression
The pETDuet-1 plasmid that builds in the step (1) is changed in the expression strain, and we have selected for use common BL21 bacterial strain promptly to obtain the proteic soluble-expression efficiently of Pcid2.It expresses flow process with common protokaryon protein expression flow process.Concise and to the point process is that expression plasmid transforms BL21 protein expression bacterial strain, chooses after 12 hours to be cloned in the 10ml Amp resistance LB liquid nutrient medium, shake bacterium about 12 hours to OD
600≈ 4, insert in the 1L Amp resistance liquid nutrient medium by 1: 100 and are cultured to OD
600≈ 1, adding 1mM IPTG induced 4 hours, 4000rpm received bacterium in centrifugal 0.5 hour, ultrasonication, and 16000rpm got supernatant in centrifugal 0.5 hour, cross the tweezer column purification, with the elution buffer wash-out that contains the 300mM imidazoles, promptly obtain having the recombinant protein of His label, the His label can remove by proteolytic cleavage, and through follow-up purifying, the product sequence and the people Pcid2 albumen that obtain are in full accord.Protein electrophoresis proves, though be at the His label that exists on the Pcid2 albumen in our purge process,, purifying comes out Dss1 albumen with Pcid2 equally, shows between the two to combine by interaction.Our experiment also shows, only expresses Pcid2 albumen and can cause this albumen to be present in the inclusion body fully, does not exist and there is soluble form.In the antibody preparation process of back, with the inclusion body protein is the antibody of antigen prepd, though may be in Western Blot level, the Pcid2 albumen of identification sex change, but in experiments such as immunoprecipitation, immunofluorescence, immunohistochemical methods, very difficult identification has the Pcid2 albumen of similar natural structure picture.Thereby our expression method has been stablized the structure of Pcid2 by the interaction of Pcid2 and its interaction protein Dss1, makes it can form correct structure picture in the expression process, thereby can exist with soluble form.Dss1 only contains 70 amino acid, it is the very little small-molecular peptides section of molecular weight, usually can exist jointly with albumen with PCI structural domain, also be called " molecular glue " by some researchist, can be unsettled zone in some protein structure, by with the interaction of Dss1, fill up unstable region, thereby make albumen form correct structure picture.Our experiment shows that on protokaryon level and eucaryon level, Dss1 can both promote the raising of Pcid2 protein stability.
Embodiment 2
(1) preparation of hybridoma
With the recombinant human Pcid2/Dss1 albumen of gained among the embodiment 1 as antigen immune Balb/c mouse (available from Beijing Vital River Experimental Animals Technology Co., Ltd.), abdominal injection, every mouse per injection 50 microgram recombinant human Pcid2/Dss1 albumen.After 2 weeks mouse is carried out immunity once more, injection volume and method are constant.Treating that mice serum is tired reaches requirement, and promptly tiring reaches more than 1: 200, prepares afterwards to carry out cytogamy, merges first three day mouse is impacted immunity.
In immune mouse, prepare murine myeloma cell Sp2/0 (ATCC CRL-1772).
The bone-marrow-derived lymphocyte and the myeloma cell Sp2/0 (ATCC CRL-1772) that will separate the sensitization that obtains from the immune mouse spleen merge (" practical immunology ", the Yang Tingbin chief editor, Changchun press, publish in December, 1994), with the Turnover of Mouse Peritoneal Macrophages is feeder cell, (purchase company with the HAT substratum in Invitrogen, HAT is xanthoglobulin (hypoxantin), aminopterin (aminopterin) and three kinds of each English lead-ins of material of Thymine deoxyriboside (thymidin) sew row, the HAT substratum just refers to contain the cell culture medium of these three kinds of materials) conventional cell cultures operation carries out selectivity and cultivates.
Then, detect above-mentioned Hybridoma Cell Culture supernatant with the ELISA method: by 96 orifice plates, 4 ℃ are spent the night with recombinant human Pcid2 albumen bag.With the phosphate buffered saline buffer washing that contains 0.05%tween-20 3 times, add supernatant 100 μ l to be checked, hatch 1h for 37 ℃.With the phosphate buffered saline buffer washing that contains 0.05%tween-20 3 times, add ELIAS secondary antibody (anti-mouse IgG-HRP) (available from Bioisystech Co., Ltd of China fir Golden Bridge in Beijing), hatch 1h for 37 ℃.Wash 3 times, add substrate developer tetramethyl benzidine (TMB) (available from Bioisystech Co., Ltd of China fir Golden Bridge in Beijing) 50 μ l, room temperature left standstill 5 minutes, added stop buffer (sulfuric acid of 2mol/L) 50 μ l.The result measures with BIORAD 680 type microplate reader, and detecting wavelength is 450nm value, the OD value be higher than negative control more than 2 times the person can be considered the positive.
Then, (limiting dilution assay is feeder cell with the Turnover of Mouse Peritoneal Macrophages) cultivated in the positive hybridoma cell cloning of selecting.Cultivate through the cloning of 2-3 wheel, obtain stable can produce the tire hybridoma cell clone of monoclonal antibody of height.With the hybridoma cell clone enlarged culturing, and frozen guarantor's kind.
A kind of positive hybridoma cell among the present invention is anti-people Pcid2 protein hybridoma cell strain 2D7-F11, with this hybridoma cell strain and be preserved in the common biological center (CGMCC of China Committee for Culture Collection of Microorganisms on June 30th, 2011, No. 3, No. 1 institute in North Star West Road, Chaoyang District, BeiJing, China city, Institute of Microorganism, Academia Sinica, 100101), preserving number is CGMCC No.5014
(2) preparation of 2D7-F11 monoclonal antibody and purifying
Above-mentioned hybridoma 2D7-F11 is seeded to the Balb/c mouse peritoneal, and preparation ascites is extracted monoclonal antibody again from ascites.The purifying of monoclonal antibody 2D7-F11: adopt Protein G affinity chromatography.At first prepare Protein G affinity column (purchasing company) in " GE ", behind PBS (phosphate buffered saline buffer) balance pillar, get the ascites that contains the 2D7-F11 monoclonal antibody and cross post, wash pillar with PBS then, approach zero to the OD value, with glycine-HCl solution (pH2.8) wash-out of 0.2M, collect elutriant, measure the OD value of each collection tube, keep the elutriant of peak region, after elutriant concentrates through dialysing-20 ℃ frozen.
Embodiment 3
The evaluation of 2D7-F11 monoclonal antibody
The 2D7-F11 monoclonal antibody that will prepare in embodiment 2 carries out western blotting to recombinant human Pcid2 albumen and human tumor cell line cell pyrolysis liquid according to a conventional method.As shown in Fig. 2,2D7-F11 monoclonal antibody and recombinant human Pcid2 albumen and the proteic cell of the natural Pcid2 of expressing human are strong positive reaction, and comprise the Dss1 no cross reaction with other albumen.
The 2D7-F11 monoclonal antibody that will prepare in embodiment 2 carries out Immunofluorescence Reactions to tumor cell line HeLa, MCF-7, HepG2 (available from the consonance cell bank) etc. according to a conventional method.The result shows that the cell of 2D7-F11 monoclonal antibody and the natural Pcid2 of expressing human is strong positive reaction, as shown in Fig. 3.
The 2D7-F11 monoclonal antibody of preparation in embodiment 2 carries out immunoprecipitation with the lysate of MCF-7 cell, the Pcid2 albumen in can the specific recognition cell pyrolysis liquid, and do not have cross reaction with other albumen, as shown in Fig. 4.
The 2D7-F11 monoclonal antibody of preparation in embodiment 2 detects the paraffin section of breast cancer tissue according to a conventional method, can observe special Pcid2 tissue distribution, as shown in Fig. 5.
Claims (9)
1. express the proteic way of soluble human source Pcid2 for one kind, it is characterized in that Pcid2 albumen and its interaction protein Dss1 are carried out coexpression, described method comprises:
(a) obtain the proteic nucleic acid sequence encoding of Pcid2 albumen and Dss1 respectively;
(b) Pcid2 albumen and the proteic nucleic acid sequence encoding of Dss1 that obtains in (a) inserted in the carrier that is fit to;
(c) express under the condition that the carrier that structure in (b) is obtained is being suitable for expressing in the host who is fit to.
2. method according to claim 1, the proteic nucleic acid sequence encoding of described people source Pcid2 is SEQ ID NO:1.
3. method according to claim 1, the proteic nucleic acid sequence encoding of described Dss1 are SEQ ID NO:2.
4. soluble recombined human Pcid2/Dss1 albumen, it is produced by each method among the claim 1-3.
5. utilize the described people of claim 4 to recombinate proteic monoclonal antibody of anti-people Pcid2 that Pcid2 albumen produces for antigen.
6. the described monoclonal antibody of claim 5 is that the mouse hybridoma cell of CGMCC No.5014 is that 2D7-F11 produces by deposit number.
7. hybridoma cell line, its deposit number is CGMCC No.5014.
8. be used to detect the test kit of Pcid2 protein content, it comprises the monoclonal antibody of claim 4 described reorganization Pcid2/Dss1 albumen and/or claim 5 or 6.
9. the application in the co-immunoprecipitation method of the immunohistochemical methods method that detects the Pcid2 tissue distribution, the immunofluorescence method that detects the Pcid2 cell distribution and detection Pcid2 interaction protein according to claim 5 or 6 described monoclonal antibodies.
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| CN201210016565.XA Active CN103215292B (en) | 2012-01-18 | 2012-01-18 | The hybridoma cell line of the solubility expression of people Pcid2 albumen and the monoclonal antibody 2D7-F11 of anti-human Pcid2 albumen and this antibody of secretion |
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| CN109913483A (en) * | 2017-12-12 | 2019-06-21 | 上海清流生物医药科技有限公司 | A kind of fermentation manufacturing technique of protein drug |
| CN110004105A (en) * | 2018-01-05 | 2019-07-12 | 上海清流生物医药科技有限公司 | A kind of application of albumen in cell culture |
| CN110882378A (en) * | 2018-09-10 | 2020-03-17 | 上海清流生物医药科技有限公司 | Application of protein in preparing medicament for preventing and treating atherosclerosis and complications |
| CN112870363A (en) * | 2021-04-03 | 2021-06-01 | 兰州大学第一医院 | Application of human PCID2 protein in preparation or screening of antitumor drugs and compound with antitumor activity |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109913483A (en) * | 2017-12-12 | 2019-06-21 | 上海清流生物医药科技有限公司 | A kind of fermentation manufacturing technique of protein drug |
| CN110004105A (en) * | 2018-01-05 | 2019-07-12 | 上海清流生物医药科技有限公司 | A kind of application of albumen in cell culture |
| CN111770988A (en) * | 2018-01-05 | 2020-10-13 | 上海清流生物医药科技有限公司 | Application of protein in cell culture |
| CN110004105B (en) * | 2018-01-05 | 2023-09-29 | 上海普佑生物医药有限公司 | Application of protein in cell culture |
| CN110882378A (en) * | 2018-09-10 | 2020-03-17 | 上海清流生物医药科技有限公司 | Application of protein in preparing medicament for preventing and treating atherosclerosis and complications |
| CN112870363A (en) * | 2021-04-03 | 2021-06-01 | 兰州大学第一医院 | Application of human PCID2 protein in preparation or screening of antitumor drugs and compound with antitumor activity |
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| CN103215292B (en) | 2015-10-07 |
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